Colebrookea oppositifolia Smith, a species used as medicinal plant in the traditional medicine of Nepal. Phytochemical screening of bioactives was performed using an integrated LC-MSn and high resolution MS (Mass Spectrometry) approach. Forty-three compounds were tentatively identified, mainly aglyconic and glycosilated flavonoids and phenolic acids, as well as other bioactives such as coumarins and terpenes were detected. Furthermore, the NF-κB and AP-1 inhibitory activity of C. oppositifolia extract were evaluated, as well as its cytotoxicity against THP-1 cells, in order to assess the potential use of this herb as a source of anti-inflammatory and cytotoxic compounds. The results so far obtained indicate that C. oppositifolia leaves extract could significantly reduce the viability of THP-1 cells (IC50 = 6.2 ± 1.2 µg/mL), as well as the activation of both NF-κB and AP-1 at the concentration of 2 μg/mL. Our results indicate that Nepalese C. oppositifolia is a valuable source of anti-inflammatory and cytotoxic compounds. The phytochemical composition reported here can partially justify the traditional uses of C. oppositifolia in Nepal, especially in the treatment of inflammatory diseases, although further research will be needed to assess the full potential of this species.Here we report the comprehensive characterization of the secondary metabolites from the leaves of  Colebrookea oppositifolia Smith is the only species of the Colebrookea genus (Lamiaceae). This plant, commonly known as “Indian squirrel tail”, is widely distributed in the subtropical regions of India, Nepal, Pakistan, Myanmar, Thailand, and China, where it grows in hills and plains at altitudes of 250–1700 m  and positive [ESI(+)] ion modes. Stationary and mobile phases, chromatographic conditions and MS parameters have been already reported in [y = 13011x – 192439 (R2 = 0.992) and y = 523050x (R2 = 0.990) for rutin in ESI(−) and ESI(+), respectively, and y = 17133.7x + 18842 (R2 = 0.997) for chlorogenic acid in ESI(−). Quantitative results were expressed as milligrams per grams of dry weight (DW).Polar constituents of COME were tentatively identified by an integrated LC-MS approach, comparing the fragmentation patterns with literature data and with standard compounds, when available. Further information was obtained searching for possible candidates in freely available MS databases by comparison of the accurate orted in . For quam/z values were obtained using a Waters Acquity H-Class UPLC system coupled to a Waters Xevo G2 QTOF MS detector, operating in both ESI(−) and ESI(+) modes. MS parameters have been already reported in [Accurate orted in , while tv/v). After 24 h incubation, the amount of metabolically active  cells was evaluated by Cell Proliferation Reagent kit WST-1  according to the manufacturer’s manual. Relative cell viability was calculated as the ratio of absorbance values of COME-treated to DMSO-only-treated cells. The IC50 value of COME was determined by four parameter logistic (4PL) analysis from the obtained viability curve. For further analysis, the concentration of COME 2 µg/mL was considered as non-toxic.The influence of COME on cell viability was determined as previously described . BrieflyEscherichia coli O111:B4 (Sigma-Aldrich) dissolved in serum-free RPMI 1640 medium (1 µg/mL). 24 h later, the activity of NF-κB/AP-1 was determined by Quanti-Blue™ medium  spectrophotometrically on FLUOstar Omega Microplate Reader  at 650 nm, according to manufacturer’s manual. Results were compared with a control incubated only with DMSO and stimulated with LPS (100% NF-κB/AP-1 activity).THP-1-XBlue™-MD2-CD14 cell line was used as a model for determination of the effect of COME on the activation of transcription factors NF-κB and AP-1 (key pro-inflammatory transcription factors). This genetically modified cell line has been specially designed for testing the activity of these transcription factors. We analyzed the ability of COME to suppress the activity of lipopolysaccharide (LPS)-activated NF-κB/AP-1 as previously described . Brieflyp < 0.05 was considered as statistically significant.All the biological experiments were performed in triplicate. The results are presented as mean values, with the error bars representing the standard error of the mean (SEM). A one-way ANOVA test was used for statistical comparisons, followed by Fisher’s LSD multiple comparison test. GraphPad Prism 8.01 software  was used to perform the statistical analysis. A value of C. oppositifolia is a shrub largely used as medicinal species in Nepal and other Asiatic countries, nevertheless, exhaustive chemical characterizations of the utilized parts are still lacking. Furthermore, despite one of the traditional uses of the leaves of this plant in Nepal and China is for the treatment of inflammatory diseases, no reports on their anti-inflammatory activity has been published up until now, nor in vitro, nor in vivo. In this article, we report for the first time the comprehensive characterization of the secondary metabolites obtained from the leaves of C. oppositifolia of Nepalese origin, and the assessment of its anti-NF-κB, anti-AP-1 and cytotoxic activities in human cell lines. Overall, the preliminary data presented here could contribute to rationalize the medicinal use of this plant by the local communities of Nepal, and could be a starting point for the isolation of potential novel anti-inflammatory and anti-tumor agents.


These unprecedented times have forced the scientific community to gather to face the COVID-19 pandemic. Efforts in diverse directions have been made. A multi-university team has focused on the identification of the host (human) proteins interacting with SARS-CoV-2 viral proteins, with the aim of hampering these interactions that may cause severe COVID-19 symptoms. Sigma-1 and sigma-2 receptors surprisingly belong to the “druggable” host proteins found, with the pan-sigma receptor modulator PB28 displaying the most potent anti–SARS-CoV-2 activity in in vitro assays. Being 20-fold more active than hydroxychloroquine, without cardiac side effects, PB28 is a promising antiviral candidate worthy of further investigation. Our research group developed PB28 in 1996 and have thoroughly characterized its biological properties since then. Structure–affinity relationship (SAfiR) studies at the sigma receptor subtypes were also undertaken with PB28 as the lead compound. We herein report our knowledge of PB28 to share information that may help to gain insight into the antiviral action of this compound and sigma receptors, while providing structural hints that may speed up the translation into therapeutics of this class of ligands. The recently obtained unique crystal structure accounts for the sigma-1 chaperone activity through polymerization–oligomerization association in which the ligand-driven conformation of the α4 helix is determinant (18F-ISO-1) . However, a recently developed class of sigma-2 antagonists is able to displace Aβ oligomers from sigma-2 neuronal receptors reducing the Aβ neurotoxic effects and shows promise in the treatment of Alzheimer’s disease . Besides the listed therapeutic potentials associated with sigma-1 and sigma-2 receptor modulation, the work by In April 2020, a multi-university team mapped the interactions between human proteins and SARS-CoV-2, with the aim of finding existing drugs that interact with these proteins and speed up the discovery of anti-SARS-CoV-2 drugs that lessen the severity of COVID-19 symptoms . Most ofin (BiP) . In addi subtype . Through subtype . Therefo subtype , Parkins subtype , and Alz subtype ; chronic subtype ; cocaine subtype . Treatme subtype . A good erminant . The leserminant . Ligandserminant . Positro disease . One of  disease   the effect on ATP cell depletion (app ratio) ≥2. A P-gp inhibitor is also able to compete with the transport of a radiolabeled P-gp substrate, but it does not induce an ATP consumption (not being transported) and has a permeability value (Papp ratio) ≤2. Results from these assays . In these experiments, PB28 significantly reduced tumor volume, to the same extent of the standard treatment (gemcitabine), and conferred a survival advantage. Importantly, no signs of toxicity  were observed in tumor-bearing and non-tumor-bearing mice under the same conditions, and the treatment was well tolerated .The widely reported role of sigma receptors in calcium signaling was also taken into account for the biological characterization of PB28 . Sigma-23H]-DTG was consistent with data obtained with the same radioligand in SK-N-SH cell homogenate . In addition, saturation of the SK-N-SH protein nuclear fraction with [3H]-DTG was consistent with data from the saturation analysis of the proteins eluted from the affinity chromatography column. This result further supported the association of PB28 with histone proteins, which are mostly expressed in the nucleosome core particle within the nucleus . Nonetheless, it was unclear if in the affinity chromatography process, the immobilized PB28 bound individual histone monomers, oligomers, or denatured forms of the nucleosome. Therefore, homology models of individual histones, or histone dimers, tetramers, and the nucleosomal core particle, were built in order to rationalize PB28 binding to these proteins , despite a high nonspecific binding , with the worst affinity recorded for the 1-naphthalenyl derivative at the sigma-1 receptor . A 10- to 50-fold reduction in the affinity was also recorded for the alkyl derivatives  at the sigma-1 receptor, whereas the drop was more drastic for the sigma-2 receptor with the methyl- and benzyl-containing analogs displaying the highest Ki values .Based on the PB28 structure, a number of structure–affinity relationship studies were undertaken with the synthesis of diverse classes of ligands. The cyclohexyl group at the piperazine nucleus was removed or replaced with other alkyl or aryl substituents. Removal of the cyclohexyl ring  as well Ki values ranging from 7.9 to 21.1 nM). By contrast, in the longer chain derivatives, an important drop in the affinity was recorded only for the hexamethylene-bearing compound at the sigma-2 receptor (Ki = 103 nM), while a low nanomolar or subnanomolar affinity was kept at the sigma-1 subtype. In particular, the compound bearing a 5-methylenes chain displayed affinity values comparable to PB28 at both subtypes. The important lipophilicity of PB28 (logP = 5.08) can be one of the causes of the high nonspecific binding in the brain of 11C-PB28 radiotracer (Kis ranging from 3.90 to 23.9 nM) and more appropriate lipophilic values were obtained, despite a 10- to 60- fold reduction of the affinity compared to PB28. Because PB28 was first synthesized as a racemic mixture, the two enantiomers were developed showing no enantioselectivity for both sigma receptor subtypes  or completely removed . Shorteriotracer . Thus, wiotracer , an aminiotracer . Compounsubtypes . The lacsubtypes , and frosubtypes . The chisubtypes . The samsubtypes . These cKi = 0.035 nM) , whereas the 7-OCH3 was particularly detrimental for the sigma-1 subtype (Ki = 9.04 nM). By contrast, the same changes in the naphthalene ring led to a subnanomolar sigma-1 affinity with the 7-methoxy substituent. On the basis of these results, a diverse series of sigma-1 selective ligands were developed as 6-methoxy-tetralin and 6-methoxy-naphthalene derivatives , in contrast to its tetralin counterpart.The methoxy group was unimportant in terms of sigma receptor affinity, as either on the tetralin or on the naphthalene nucleus the absence of the methoxy group was not detrimental for both subtypes . Notewor.035 nM) . On the ivatives . In addiKis = 143 nM, and >5,000 nM, respectively), excellent sigma-1 affinity values  were obtained when the basicity of the distal N-atom was kept as in PB28 analogues bearing 1-cyclohexylpiperidine and 1-propionyl-4-cyclohexylpiperazine. In particular, the latter compound displayed an excellent profile as a sigma-1 selective ligand with its subnanomolar sigma-1 affinity and a 1600-fold selectivity against the sigma-2 subtype. The same conclusion could be drawn from the quaternarization of the piperazine N-atoms in the corresponding methylammonium salts: the “distal” N-atom lone pair must be available for interaction with the sigma-1 receptor, otherwise resulting in a decreased affinity (Ki = 338 nM). On the other hand, quaternarization of one of the N-atoms led to a just modest (10-fold) drop in the affinity for the sigma-2 subtype, supporting an ionic interaction of the ligands at the sigma-2 binding site  but a notable sigma-2 receptor affinity (Ki = 12.6 nM). On the other hand, the less bulky N-indolyl (with one fused benzene ring less compared to the carbazole) had similar single-digit nanomolar affinity at both sigma subtypes . These data together suggest a less tolerant region for hydrophobic substituents in the sigma-1 compared to the sigma-2 subtype binding pocket (Kis from 2 to 26.6 nM ranges) (Kis = from 6.50 to 28.9 nM) (Kis ranging from 0.25 to 45.5 nM)  . The dat28.9 nM) . Replace45.5 nM) . Of inteaffinity . PB28 coAs for the changes on the cyclohexylpiperazine basic moiety with other moieties, the only result noteworthy was the affinity conferred by the 6,7-dimethoxytetrahydroisoquinoline. The presence of the latter is consistently strongly detrimental for the sigma-1 binding, whereas low nanomolar affinity values for the sigma-2 receptor are maintained when the basic moiety is linked to hydrophobic portions .PB28-based fluorescent tracers: Given the need of biological tools to study the “enigmatic” sigma receptors, the PB28 structure was modified with the aim of obtaining fluorescent tracers. Previous structural changes allowed one to identify the position that could be modified without an important decrease in sigma receptor affinity phthalimide  (affinity . The hig 6.9 nM) . This gr 6.9 nM) . Importa 6.9 nM) . In orde 6.9 nM)  (Pati et 6.9 nM) .According to a multitarget ligands approach, the PB28 structure has also been modified in order to incorporate metal chelating portions and/or P-gp interacting moieties in order to increase the antitumor potentials with encouraging results, in particular from pancreatic tumors .We have herein presented the results obtained during the years with our lead compound and reference sigma receptor modulator PB28. Since PB28 binds equally well the sigma receptor subtypes (with subnanomolar affinity), it is important to elucidate whether its anti–SARS-CoV-2 activity is due to the interaction with one of the two subtypes, or both, and what interaction type is needed (agonist versus antagonist), although an off-target effect cannot be ruled out. Herein, we share our knowledge about PB28 pharmacology and provide structural elements that may help to drive future modifications on PB28 structure to optimize it toward a therapeutic drug. Additionally, we provide information about the structure–affinity relationship studies that have led to the generation of several PB28 derivatives, some of which display high nanomolar affinity and selectivity toward one of the two subtypes. The availability of these small molecules, together with PB28-based fluorescent tracers may be of further help to the scientific community working against the COVID-19 pandemic to better define the sigma receptor subtypes involvement in the antiviral action and quickly move forward. It is worth noting that the chaperone nature of the sigma-1 receptor  often makes the activity of the sigma receptor ligands cell-dependent. Therefore, we can expect that the affinity values of sigma receptor ligands do not necessarily reflect the antiviral activity. Therefore, structure antiviral-activity studies with the molecules herein presented and other molecules will be of extreme aid to formulate hypotheses about the sigma receptor involvement in the action against SARS-CoV-2. The results will possibly shed light on the mechanisms of interaction that do not only involve sigma receptor affinity, but also take into account the possible sigma receptor client proteins.CA designed and drafted this work, NC and FB critically revised it for intellectual content, MN and MC contributed to conception and analyzed and interpreted the biological sections, and FA contributed to collection of data and revisions.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The likelihood of inpatient mortality has been found to be reduced by increased nurse staffing in several settings, including general wards, emergency departments, and intensive care units. However, less research has investigated cases where patients die in the community setting due to a health problem that occurred after they were discharged post-surgery, because it is difficult to integrate hospital data and local community data. Therefore, this study investigated the association between the bed-to-nurse ratio and 30-day post-discharge mortality in patients undergoing surgery using national administrative data.The study analyzed data from 129,923 patients who underwent surgery between January 2014 and December 2015. The bed-to-nurse ratio was categorized as level 1 (less than 2.5), level 2 (2.5–3.4), level 3 (3.5–4.4), and level 4 (4.5 or greater). The chi-square test and GEE logistic regression analyses were used to explore the association between the bed-to-nurse ratio and 30-day post-discharge mortality.1355 (0.01%) patients died within 30 days post-discharge. The 30-day post-discharge mortality rate in hospitals with a level 4 was 2.5%, representing a statistically significant difference from the rates of 0.8, 2 and 1.8% in hospitals with level 1, level 2, and level 3 staffing, respectively. In addition, the death rate was significantly lower at hospitals with a level 1 (OR = 0.62) or level 2 (OR = 0.63) bed-to-nurse ratio, using level 4 as reference.The results of this study are highly meaningful in that they underscore the necessity of in-hospital discharge nursing and continued post-discharge nursing care as a way to reduce post-discharge mortality risk. Furthermore, the relationship between nurse staffing levels and 30-day post-discharge mortality implies the need for a greater focus on discharge education. Policies are required to achieve proper nurse staffing levels in Korea, and thereby to enhance patient outcomes. Surgery is an invasive process in which various human body parts undergo incision, removal, prosthesis application, and/or suturing, depending on the procedure. Therefore, surgery is accompanied by a high risk of complications such as gastrointestinal problems and complications, wound disclosure and surgical infection, venous thromboembolism, and failure to thrive or malnutrition. Due to the high rate of postoperative complications, 11.3–11.9% of patients who have undergone general surgery, craniotomy, or vascular surgery are readmitted within 30 days after discharge .Studies of the associations between inpatient or post-discharge patient mortality and nurse staffing for patients with various conditions—treated medically or surgically—have been conducted in many countries, and the likelihood of patient mortality has been found to be reduced by increased nurse staffing in several settings, including general wards, emergency departments, and intensive care units (ICU) –8. FurthThe goal of nursing is to conduct safe and high-quality care for patients, and discharge education for self-care of patients at home after discharge is a core nursing process. That is why insufficient discharge preparation by nurses during admission makes it difficult for patients and their families to carry out treatment instructions and self-care for surgical wounds, and to find and link appropriate supporting resources , and canThis was a cross-sectional study using a secondary analysis of National Health Insurance Service (NHIS) data from January 2014 to December 2015 to examine the association between the bed-to-nurse ratio and death within 30 days after discharge among patients who underwent surgery.This cross-sectional, retrospective study was performed using NHIS data on 129,923 patients. The NHIS is a nationwide, comprehensive health insurance system that is administered by the government of Korea. The NHIS manages all fee-for-service claim data, which encompass patients’ medical history from all medical institutions. We extracted patient-level and hospital-level data for this study. The inclusion criteria for patients in this study were patients aged between 20 and 85 who underwent 12 types of surgery between January 2014 and December 2015. The 12 types of surgery analyzed in this study were determined using Korean Diagnosis-Related Group (KDRG) categories with relatively high death rates according to previous studies . Their KDRG categories were as follows: major craniotomy except for trauma (B011-B019), other craniotomy except for trauma (B021-B022), craniotomy for trauma (B030), cardiac valve procedure with cardiac catheter (F021-F023), cardiac valve procedure without cardiac catheter (F031-F033), coronary bypass with/without cardiac catheter (F041-F044), major reconstructive vascular procedure (F061-F064), amputation for circulatory system disorders (F120), esophageal procedure (G011-G013), major small and large bowel procedure (G031-G033), stomach and duodenal procedure (G041-G046), and pancreas, liver and shunt procedure (H011-H016) . The excOverall, 129,923 surgical patients from 214 hospitals were included in the analysis, after the further exclusion of 4822 patients who died in-hospital, 1663 patients who were readmitted within 30 days, and 1972 patients who underwent surgery at small hospitals with less than the minimum patient volume (fewer than 24 discharged patients during the study period) were then excluded to obtain reliable estimates from the statistical analysis , 18. TheThe outcome variable was death within 30 days after discharge to home from a hospital. The death was confirmed by integrating the data of 129,923 patients extracted from the NHIS data and death registry data of Statistics Korea using temporary IDs.The bed-to-nurse ratio of the general wards, the primary independent variable of interest, calculated by the number of total general ward beds divided by the number of nurses participating in the nursing care of the patients using those beds . In thisThe following variables related to characteristics of hospitals were controlled for: hospital type  based on medical laws, hospital ownership , hospital location , and bed-to-physician ratio.The following additional patient-related variables were controlled for: age, gender, complications or comorbidities (CCs), type of health insurance , and admission route . CCs were classified as 0 (no CCs), 1 (minor CCs), 2 (moderate CCs), and 3 (severe CCs) based on the KDRG Grouper, applying the complication and comorbidity levels (CCLs) of the Australian Refined Diagnosis Related Groups (ARDRGs). The CCLs provide a weighting of case complexity using integer values between 0 and 4 for all diagnoses; they make it possible to consider both already-present diagnoses at the time of admission (comorbidities) and acquired diagnoses after the patient’s admission (complications) when evaluating post-discharge mortality .χ2 test was used to assess differences in categorical variables . Generalized estimating equation (GEE) logistic regression was used to calculate the adjusted odds ratios (ORs) for 30-day post-discharge mortality, controlling for confounding variables . As there was a correlation between subjects who treated in the same hospital due to the information about the hospital being the same, GEE logistic regression was used, because it has the advantage of correcting for the standard error when estimating parameters. Data were analyzed using SAS 9.4 . Significance was set at P < .05.In the statistical analyses, the The characteristics of the hospitals included in this study are presented in Table χ2 = 43.33, P < .001), hospital ownership , hospital location , bed-to-physician ratio , and the bed-to-nurse ratio of general wards  , gender , type of surgery , CCs , type of health insurance , and admission route   for post-discharge mortality, as shown in Table For every 1-year increase in patients’ age, the mortality rate was found to increase by 4% . Males had a significantly higher risk of mortality than females, with an OR of 1.40 (95% CI = 1.22–1.60). The ORs showed statistically significant differences depending on surgery type. In terms of type of surgery, patients who underwent major craniotomy except for trauma were found to have an OR for mortality of 1.66 (95% CI = 1.21–2.28). Other craniotomy except for trauma was associated with an OR of 1.62 (95% CI = 1.15–2.27), major reconstructive vascular procedure with OR of 2.40 (95% CI = 1.46–3.93), major small and large bowel procedure with an OR of 0.42 (95% CI = 0.29–0.62), and stomach and duodenal procedure with an OR of 0.70 (95% CI = 0.53–0.93), using pancreas, liver and shunt procedures as the reference category.Patient with no CCs were found to have an OR for mortality of 0.38 (95% CI = 0.26–0.55) and those with moderate CCs had an OR of 1.57 (95% CI = 1.12–2.19) compared to patients with severe CC. In terms of type of health insurance, self-employed beneficiaries had an OR for mortality of 0.53 (95% CI = 0.32–0.89), and employed beneficiaries had an OR of 0.43 (95% CI = 0.25–0.72) compared to Medical Aid dependents. The OR for the emergency department admission route was 2.73 (95% CI = 2.33–3.21) compared to admission via an outpatient department.The purpose of the present study was to investigate the association between nurse staffing levels of hospitals and 30-day mortality post-discharge in Korea. The study showed that 1355 patients died within 30 days after discharge, and that higher nurse staffing levels were associated with significantly lower ORs for mortality. More specifically, bed-to-nurse ratios of less than 2.5 and 2.5–3.4 were associated with 30-day post-discharge mortality probabilities that were 38%  and 37%  lower, respectively, than that of a bed-to-nurse ratio of 4.5 or over. This result is similar to that of a previous study conducted in hospitals in Korea, which showed that when the nurse staffing standard of the Adjusted Differentiated Inpatient Nursing Fees by staffing grades of the NHIS (the same as used in this study) was applied, patients who underwent surgery in hospitals with a nurse staffing grade of 2–3 (bed-to-nurse ratio 2.5–3.4), 4–5 (bed-to-nurse ratio 3.5–4.4), and 6–7 (bed-to-nurse ratio 4.5 or greater) had 57, 78, and 199% higher likelihoods of in-hospital mortality, respectively, than patients who underwent surgery in hospitals with a nurse staffing grade of 0–1 (bed-to-nurse ratio less than 2.5) . On thisIn this study, nurse staffing was the only hospital-related variable that showed a significant association with post-discharge mortality of surgical patients. Although it is difficult to find studies that allow direct comparisons with the present study, as not many studies have been conducted on the association between nurse staffing and post-discharge mortality, the results of this study are similar to those of studies that analyzed in-hospital or 30-day mortality. In a study conducted among patients who received treatment in an ICU due to cardiovascular disease, when the ICU staffing level of general hospitals gradually increased from 0.5 or below to 2.0 or above, 30-day mortality after discharge increased significantly by 37–50% . In a stIn previous studies, the variable that mediated the relationship between nurse staffing and the patient outcome of death was inferred to be an excessive workload and missed nursing care , 11, in Although the nurse-to-patient ratio is an important variable affecting patient mortality, the requirement of two nurses per five patients specified in the Medical Service Act of Korea is a relatively low nurse staffing standard compared to other countries . MoreoveThis study classified the bed-to-nurse ratio by applying the Adjusted Differentiated Inpatient Nursing Fees by staffing grades, which sets the rate of operated sickbeds and nurses using an arithmetical standard. However, in other countries, research has been conducted based on the nursing skill mix. In a study relating to 30-day mortality after heart surgery, when the proportion of nurses with less than 2 years of ICU experience among nurses who worked in the ICU was over 1/3, mortality increased by 18% . AdditioBased on the issues discussed above, this study has the following implications. There is a need for research relating to economic analyses, such as ensuring appropriate nurse staffing levels and cost-benefit analyses of nursing-led discharge management programs, in order to reduce in-hospital and post-discharge mortality. Additionally, when making policy decisions, the period in which benefits occur should be extended after discharge instead of being limited to the hospital stay to obtain accurate and valid findings.The major results related to the independent variables analyzed in this study are as follows. The variables that were significantly associated with 30-day post-discharge mortality were primarily general characteristics of the patients, and hospital characteristics—except for nurse staffing—did not show significant associations. These results are similar to those of a study that analyzed inpatient mortality . The resIn a previous study, age was found to be associated with post-surgical mortality , 17, andIn this study, moderate complications or comorbidities (CCs) were associated with a 57% higher risk of mortality than severe CCs. This is similar to the result of a previous Korean study on post-surgical inpatient mortality and complications, which found that moderate CCs were associated with an elevated risk . In termWhen the admission route was the emergency department, both post-discharge mortality and in-hospital mortality have been found to be higher , 10, 17,The major limitation of this study is related to the measurement of nurse staffing levels. International studies have measured nurse staffing levels in various ways, such nursing hours per patient day, nurse staffing by shift or the occupancy rate, and the education level of nurses , 23, 32.This study investigated the association between nurse staffing in Korean hospitals and the mortality risk of patients who had been discharged after receiving surgery and were not readmitted. Patients were analyzed by linking National Health Insurance data with the annual death statistics from Statistics Korea. This study has significant meaning in that it confirmed that an adequate nurse staffing level was significantly associated with 30-day post-discharge mortality among surgical patients after controlling for hospital characteristics.Additional file 1: Supplement 1. Flow chart of patient selection based on the National Health Insurance Service data.


Chronic inflammation is involved in the formation and enlargement of cerebral aneurysms (CAs), with macrophages playing a key role in the process. The present study evaluated visualization of macrophages present in CAs using an activatable fluorescent probe (IONP-ICG) comprising an iron oxide nanoparticles (IONPs) conjugated with indocyanine green (ICG).n = 8), and ex vivo near-infrared fluorescence (NIRF) imaging and histological assessment of exposed CAs and cerebral arteries were performed 48 h later. Similar evaluations were performed in the control group, which included CA model rats given IONPs or ICG (n = 8 each).IONP-ICG was intravenously administered to 15-week-old CA model rats (P = 0.037). Moreover, all 11 CAs that did not exhibit NIRF signals were iron-negative, while the two CAs that exhibited NIRF signals were both iron-positive (P = 0.013).ICG-derived NIRF signals were detected in three IONP-ICG group rats but not in IONP or ICG control groups. Among the three rats that exhibited signals, NIRF signal accumulation was observed in the CA of two rats and at the site of hemodynamic stress in the left posterior cerebral artery in one rat. Histologically, NIRF signals correlated strongly with macrophage localization. A total of 13 CAs formed in the IONP-ICG group. The number of macrophages in the CA wall was significantly greater in the two CAs that exhibited NIRF signals compared to the remaining 11 CAs that did not (NIRF imaging using an activatable IONP-ICG probe is feasible for detecting the macrophage-rich regions in CAs and the cerebral artery wall, which is considered an early lesion in the process of CA formation. Chronic inflammation is involved in the formation and enlargement of cerebral aneurysms (CAs), and macrophages, which are the primary inflammatory cells that accumulate in the aneurysm wall, play an essential role in these processes . Moreovein vivo due to their high biocompatibility and low toxicity (Iron oxide nanoparticles (IONPs) have been widely utilized toxicity , and thetoxicity . Since itoxicity . This prtoxicity , is the in vitro and intracellular activation of NIRF. Furthermore, IONP-ICG synthesized by mixing with a molecular ratio of 1:20 IONP:ICG enabled NIRF imaging of macrophages in atherosclerotic plaques with the greatest background ratio. Therefore, we postulated that NIRF imaging using an IONP-ICG probe might enable targeted visualization of CA macrophages, facilitating prediction of the formation, enlargement, and rupture of CAs. The present study evaluated whether macrophages localized to CAs can be specifically detected using our activatable IONP-ICG NIRF probe.When multiple fluorescent molecules are in proximity to each other, their interaction leads to fluorescence self-quenching . Moreove®-D-spio; diameter, 20 nm; mean molecular weight, 3500 kDa; amino groups on particle surface) were purchased from Corefront Co. . ICG-EG4-Sulfo-OSu was purchased from Dojindo Molecular Technologies . Methoxypolyethylene glycol (PEG) succinate N-hydroxysuccinimide (NHS)  was purchased from NOF America Co. . ICG was purchased from Tokyo Chemical Industry Co., Ltd. .IONPs coated with dextran  and ICG-EG4-Sulfo-OSu (8.5 mmol/L in dimethyl sulfoxide) in a 1:20 IONP:ICG molecular ratio . The concentration of IONPs and ICG in synthesized IONP-ICG was determined by measuring the absorption at 535 and 768 nm using a Shimadzu UV-Vis NIR system  to determine the number of ICG molecules conjugated to each IONP. As a control probe, IONP-PEG was synthesized by mixing the IONP nanomag®-D-spio  and PEG-NHS-ester  to quench the amino groups of the particles.Synthesis of IONP-ICG was performed according to our previous report . Brieflyn = 8) were intravenously injected with IONP-ICG  via the tail vein. The mean number of ICG molecules conjugated to each IONP in IONP-ICG was 7.5 ± 0.6 in the present study. Control group rats were injected with either IONP-PEG  or ICG . In total, 24 rats were used in the present study (n = 8 per group).All animal experiments were conducted following institutional guidelines and were approved by the Kyoto University Animal Care Committee. CA model rats were prepared as described previously , with so2/sr) of each region of interest after subtraction of background fluorescence was measured.48 h after probe administration, rats were deeply anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital and perfused transcardially with 4% paraformaldehyde after 500 μL of blood was collected from the heart to measure the fluorescence intensity. After euthanasia by cervical dislocation and decapitation, the brain along with cerebral arteries, liver, spleen, kidneys, lungs, heart, and muscles were dissected. Cerebral arteries were stripped from the brain after the microscopic observation described below. Fluorescence images of tissues were acquired using an IVIS Imaging System 200  to determine the distribution of ICG. Similar-sized regions of interest were circled within respective tissues, and the mean fluorescence intensity  mounted on an MVX10 macro zoom fluorescence stereomicroscope  equipped with a Cy7 filter set . NIRF images of the brain were acquired by multispectral imaging at 10-nm wavelength increments from 770 to 950 nm with an exposure time of 5 s. Nuance version 3.0.2 software (PerkinElmer) was used to examine the fluorescence signals derived from ICG by measuring the fluorescence spectra, unmixing the autofluorescence spectra of the tissues, and eliminating background noise. Visible light and NIRF imaging were performed from the ventral side of the brain. Cerebral arteries as well as whole circle of Willis arterial rings stripped from the brain underwent visible light and NIRF imaging.Excised brains were washed with 0.04% bromophenol blue solution to facilitate visualization of cerebral arteries by visible light imaging then immediately washed with phosphate-buffered saline. After, brains were imaged Cerebral aneurysm was defined as bulging or saccular shape of the cerebral artery wall with disruption of the internal elastic lamina. CAs were searched by stereomicroscopy from the ventral side of the brain and from both ventral and dorsal sides of cerebral arteries stripped from the brain. Bifurcations of the right anterior cerebral and olfactory arteries, including the CAs, as well as stereomicroscopy-detected CAs in the area around the circle of Willis arterial rings were carefully excised. Two or more CAs arising side-by-side at the same location were evaluated separately. In addition, if CAs could not be detected by stereomicroscopy, arteries showing NIRF signals derived from ICG were still excised. Cryosections  were obtained from freshly frozen samples. ICG NIRF signals were observed using a BZ-X710 fluorescence microscope  equipped with a filter for ICG . Immunohistological staining was then performed using an anti-mouse ionized calcium binding adapter molecule 1 (Iba1) antibody  for localization of macrophages. Iron staining was performed using a Berlin blue staining set . Elastica van Gieson (EVG) staining was performed to assess disruption of the internal elastic lamina. Light microscopy was performed using an FSX100 microscope (Olympus).Morphological evaluation of CAs was performed as described previously , with soU-test between two groups and Kruskal-Wallis test followed by the Steel-Dwass test between three groups or more were used for continuous variables. A P < 0.05 was considered statistically significant.Statistical analyses were performed using JMP version 11 software . Quantitative data were expressed as the mean ± standard deviation. Fisher’s exact test between two groups and Pearson’s Chi-square test between three groups were used to examine categorical variables. The Mann-Whitney P < 0.05). In the IONP-PEG group, the NIRF signal was highest in the kidney, with barely any detected in other organs. In the ICG group, the NIRF signal was most intense in the liver followed by the kidney; accumulation was negligible in other organs. Comparison of each organ between the three groups revealed significantly greater NIRF signal accumulation in the IONP-ICG compared to IONP-PEG and ICG groups for all organs except the blood (P < 0.001). NIRF signal intensities in the blood were extremely low in all three groups and did not differ significantly between them (P = 0.11).The results of biodistribution studies 48 h after probe administration are summarized in ICG-derived NIRF signals were detected in the CA or cerebral artery of three IONP-ICG rats by NIRF imaging. In contrast, ICG-derived NIRF signals were not detected in IONP-PEG or ICG groups. Microscopic and histological findings detected CAs in all 24 rats included in the study, amounting to 38 total CAs. Of them, 13 were in the IONP-ICG group, 13 in the IONP-PEG, and 12 in the ICG group. P = 0.0013). Mean CA wall thickness was 17.2 ± 22.7 μm  overall, with 16.8 ± 5.6 μm  in the IONP-ICG group, 22.5 ± 33.8 μm  in the IONP-PEG group, and 11.5 ± 3.5 μm  in the ICG group. One saccular CA in the left posterior cerebral artery in the IONP-PEG group did not have a lumen, preventing measurement of CA wall thickness. Therefore, this CA was excluded from the calculation of overall mean CA wall thickness. The mean wall thickness of three saccular CAs of the left posterior cerebral artery was 88.7 ± 30.0 μm , which is significantly thicker than that of bulging CAs .Mean CA size was 150 ± 190 μm  overall, with 130 ± 176 μm  in the IONP-ICG group, 208 ± 257 μm  in the IONP-PEG group, and 101 ± 60 μm  in the ICG group. The mean size of the four saccular CAs at the left posterior cerebral artery was 573 ± 366 μm , which was significantly greater than that of bulging CAs . An iron-positive wall was observed in five CAs (13%) overall, with two (15%) in the IONP-ICG group, three (23%) in the IONP-PEG, and 0 (0%) in the ICG group. All four saccular CAs of the left posterior cerebral artery were iron-positive. None of the items listed in P = 0.99; number of saccular CAs, P = 0.16; CA size, P = 0.15; CA wall thickness, P = 0.98; number of macrophages, P = 0.13; iron positivity, P = 0.22).The mean number of macrophages was 6.6 ± 7.5  overall, with 5.8 ± 6.3  in the IONP-ICG group, 10.0 ± 2.8  in the IONP-PEG group, and 3.8 ± 2.0  in the ICG group. The mean number of macrophages in the four saccular CAs of the left posterior cerebral artery was 24.3 ± 8.0 , which was significantly greater than that of bulging CAs . The mean wall thickness of CAs with and without NIRF signals was 49.0 μm  and 10.9 ± 3.9 μm , respectively; this difference was not significant (P = 0.092). On the other hand, the mean number of macrophages in CAs with NIRF signals  was significantly different from those without signals . Furthermore, both CAs with NIRF signals showed iron-positive staining, while all 11 CAs without signals were iron-negative, indicating that CAs with NIRF signals were significantly more likely to be iron-positive (P = 0.013).To determine the type of CAs that exhibited NIRF signals, the two CAs in the IONP-ICG group with NIRF signals were compared with the 11 CAs of this group without signals . AlthougUsing an activatable IONP-ICG NIRF probe, the present study successfully detected localized macrophage-rich CA-associated chronic inflammation in rats. Specifically, histological findings showed a strong correlation between NIRF signals in the CA wall and macrophages-rich regions. Furthermore, iron positivity in the same area indicates that NIRF signals were derived from the IONP-ICG administered to live rats. In other words, intravenously administered IONP-ICG was taken up by macrophages accumulating in the CA wall, leading to activation of quenched IONP-ICG fluorescence. Thus, we concluded that macrophage localization can be determined in CAs through NIRF imaging.The quenching of fluorescence probably occurred via the interaction between ICG and IONP (energy transfer) . AlthougAs mentioned above, IONPs decrease magnetic resonance imaging signals in the aneurysm wall when taken up by macrophages . A previin vitro experiment demonstrating that divergent differentiation of macrophages into M1 or M2 subsets does not affect uptake of IONPs  and humans (2.5–5 mg ferumoxytol/kg) , indicating the necessity for caution when interpreting statistical results.Several limitations to this study must be considered. First, CAs were examined ckground  and the ckground , NIRF siytol/kg) . Furtherkinetics , determiIn the present study, we demonstrated the potential of an activatable IONP-ICG NIRF probe for imaging of macrophages that accumulate in CAs. The individual reagents used  are highly biocompatible and approved for clinical use by the United States Food and Drug Administration  indicatiUsing activatable IONP-ICG NIRF imaging, we for the first time succeeded in detecting the macrophage-rich regions in CAs and/or the cerebral artery wall, which is considered an early lesion in the process of CA development.rocky@kuhp.kyoto-u.ac.jp.The datasets analyzed in this manuscript are not publicly available. Requests to access the datasets should be directed to The animal study was reviewed and approved by Kyoto University Animal Care Committee.HI, AI, and KS conceived and designed the study. HI and KS performed the experiments and wrote the manuscript. HI analyzed the data. All authors read, reviewed, edited and approved the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HTR2A) genetic polymorphisms. By using gene-based association analysis, we hope to identify genetic variants of HTR2A which are related to MDD susceptibility and its antidepressant therapeutic response. 288 HTR2A single nucleotide polymorphisms in MDD susceptibility have been investigated through a case–control  study, as well as in antidepressant efficacy (n = 455) in our current research. The 21-item Hamilton Rating Scale for Depression was used to evaluate measures of antidepressant therapeutic efficacy. From two MDD groups in the antidepressant therapeutic response, by using gene-based analyses, we have identified 14 polymorphisms as suggestive markers for therapeutic response (13 for remission and 1 for response) in both meta- and mega-analyses. All of these HTR2A reported polymorphisms did not reach statistical significance in the case–control association study. This current investigation supported the link between HTR2A variants and antidepressant therapeutic response in MDD but not with MDD susceptibility.The serotonin [5-hydroxytryptamine (5-HT)] system has been implicated in the pathogenesis of major depressive disorder (MDD). Among the 5-HT receptor subtypes, 5-HT2 is one of the major pharmacological therapeutic targets for MDD. There have been inconsistent findings in previous pharmacogenetic studies investigating the antidepressant therapeutic response using one or several 5-HT2A ( One of the common and sometimes fatal mental disorders is major depressive disorder (MDD), which is also a leading cause of disability worldwide . Even wiHTR2A) gene is said to be correlated to susceptibility in MDD  of the HTR2A gene located on 13q14.2 are A-1438G (rs6311) and T102C (rs6313) polymorphisms. Initially, Zhang et al. proposed that MDD is associated with the C allele of the rs6313 polymorphism in 1997 , rs6313 (T > C), and rs7997012   . The mosdication . Within dication . Lin et is study . In addiis study .HTR2A genetic studies in MDD susceptibility and therapeutic response should be done with particular care, with regard to the complex structure of the gene, and the fact that most studies explored effects of one or several SNPs. It is possible that MDD susceptibility or antidepressant therapeutic responses may be affected by other variants in the HTR2A gene. Genes which may increase the vulnerability to complex diseases and therapeutic responses to medication may be detected using gene-based association tests. These tests can identify genes with genome-wide significance, but no single SNP effect is able to give genome-wide significance by univariate tests. Thus, the hypothesis that HTR2A may influence MDD susceptibility and antidepressant therapeutic response is verified with gene-based analysis of genome-wide data. The present study is a secondary analysis of previously published international genome-wide association study (GWAS) data  data . Only Tan = 162), citalopram (n = 20), escitalopram (n = 162), and fluoxetine (n = 77). Only three subjects were treated with sertraline, and four subjects were treated with non-SSRI (venlafaxine) and hence were excluded from the analyses to eliminate the difference from the effects of sertraline or venlafaxine. Depression severity was measured by asking all participants to answer the 21-item questionnaires of the HRSD. Patients were repetitively assessed at baseline and weeks 2, 4, and 8.A total of 455 patients were recruited in the study, who were diagnosed with MDD in two central hospitals in Taiwan, with 187 from TVGM  and 268 from NHRI  . InclusiRandom selection of healthy controls from a pool of 3,380 healthy individuals recruited from community subjects of the TWB with Taiwanese ancestry was made. These selected individuals were used for a case–control association study. The recruitment details were documented elsewhere . In shorExperiments were conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of Taipei Veterans General Hospital (VGHIRB No.: 2014-06-001B). Written informed consent forms were obtained from all participants ensuring adequate understanding of the study.Four repeated phenotypes were used to measure the different treatment responses, including stem-depressed, remitted, and response. 1) Initially, the sum score of the 21-item HRSD at week 8 was calculated and recorded as 1 if the sum score is greater than 7 and recorded as 2  otherwise. 2) We then calculated the HRSD percentage change  at week 4 and recorded as non-response if the percentage change is less than 50% reduction in symptoms and recorded as response if otherwise. 3) We also used the percentage change of the HRSD (continuous variable) as a treatment response index. 4) For stem-depressed phenotype analysis, “depressed mood” was measured using the first item of the HRSD at week 4 and recorded as 1 if the score exceeds or is equal to 3  and recorded as 2 otherwise. Refer to Lin et al. for detailed definitions of the treatment response phenotypes .Multiple imputation methods were used to solve the problem of missing phenotypes . Multiplp-value > 0.0001), genotype missing rate (≤5%), minor allele frequency (MAF ≥ 0.05), and quality in clustering  were performed to the GWAS SNP data. As a result, a total of 647,030 SNPs in the samples were retained for imputation. The genotyping call rate was 99.9% for all subjects. The genotyped markers from SNP arrays were used to impute genotypes which were not observed using IMPUTE2 v3 (https://mathgen.stats.ox.ac.uk/impute/data_download_1000G_phase1_integrated_SHAPEIT2.html). Only high genotype information content SNPs that were imputed  were included in association analyses. In total, 30,040,257 SNPs were imputed with high confidence for each individual in the samples. A total of 4,241,701 SNPs were retained for analyses following the same quality control procedures for markers. All of the 4,241,701 SNPs were mapped into 22,609 protein-coding genes using 50 kb upstream and downstream of the gene boundaries. Selection of the HTR2A gene variants was made, and these were mapped from the imputed genome-wide association SNP data. As a result, a total of 288 SNPs were mapped in the HTR2A gene.All participants were genotyped using Illumina HumanOmniExpressExome BeadChips in the International selective serotonin reuptake inhibitors (SSRI) Pharmacogenomics Consortium. A total of 455 subjects were genotyped with 951,123 SNPs. Quality control procedures were done firstly with each individual, including sample quality, kinship, and population stratification. Initially, plate-wise genotyping biases were checked. Samples with a plate pass rate greater than 97% were retained in the analysis. A total of 18 samples (11 from NHRI and 7 from TVGM) were removed during this step. Second, we checked inbreeding coefficient and identity by state so that samples with strong kinship were eliminated. A total of 9 individuals (4 from NRHI and 5 from TVGM) with measures far away from clustering were removed from the samples . Third, PUTE2 v3 , with ha., NHRI and TVGM) was conducted using Student’s t-test (for continuous data) and the chi-square test . The difference test of treatment response phenotypes between treatment responders and nonresponders were conducted using Student’s t-test (for continuous data) and the chi-square test .A difference test of demographic and clinical characteristics between two independent samples  within a gene. Only SNPs having r2 < 0.5 with each other were retained for each gene in order to account for linkage disequilibrium (LD) among markers. Gene-based association tests were performed by fitting both the logistic regression and linear models. We summed the association signal from all SNPs that passed the above criteria within a gene and corrected the sum for LD to generate a test statistic. An empirical gene-based p-value was calculated with 50,000 permutation tests within each gene.Gene-based association analyses were conducted to obtain gene-level empirical significance estimation. We collected information from a set of SNPs  were combined and summarized using the inverse gamma model with a shape parameter (α) of 1 (that is the Fisher exact method) , and genes, with p-values less than 1 × 10–2 (or 5 × 10–2), in single-marker and gene-based association analysis, either in mega-analysis  or meta-analysis , and considered to be significant (or suggestive).Association results across two independent samples (i.e method) . We follfindings . We repoAll analyses were conducted with R version 3.0.2 and PLINK version 1.90b3.37 64-bit.A summary of general demography for 428 MDD patients treated with SSRI antidepressants was shown in HTR2A SNPs, 22 SNPs with odds ratios (ORs) ranged from 0.51 to 2.21 in NHRI samples and ranged from 0.73 to 2.16 in TVGM samples (0.59–2.04 in combined samples) were reported to have suggestive signals, which reached p-value < 5.0 × 10–3 either in mega-analysis or in meta-analysis (HTR2A) were significantly  associated with remission (score) in both meta-analysis and mega-analysis; one out of eight SNPs (mapped to HTR2A) was significantly associated with response  in both meta-analysis and mega-analysis; and none were significantly associated with stem-depressed (item-wise) in both meta-analysis and mega-analysis. For gene plots of four treatment response indices for mega- and meta-analysis, refer to HTR2A gene showed suggestive association  in mega-analysis and/or meta-analysis in remitted (score) and in response (%ΔHRSD).In single-marker association analyses of 288 analysis . Among tWe also conducted a case–control association study using samples from NHRI and TVGH (421 MDD patients) and TWB . All reported genes and SNPs reported did not reach a significant association level.  of the STAR*D study . FurtherHTR2A SNPs were found to be suggestive markers for treatment response phenotypes (13 for remission and one for response). Only rs977003 is a chip SNP, and the remaining 13 SNPs are imputed SNPs. In the positron emission tomography (PET) study, rs977003 appears to be significantly associated with serotonin transporter binding potential  were reported to have suggestive signals either in mega-analysis or in meta-analysis. Using samples from NHRI, TVGM, as well as a combination of both, 13 SNPs (where rs17068986 SNP was reported in both remission and response) out of the 14 otential . This pootential . TherefoHTR2A SNPs related to MDD therapeutic response, MDD patients with the rs7333412 GG genotype have been reported to be less prone to antidepressants than patients of A allele carriers  exhibited direct eQTL effects. Of note, functional annotation by HaploReg indicated that transcriptional regulation activity exists at the seven SNP loci for the HTR2A gene in peripheral blood monocytes, dendritic cells, prefrontal cortex, blood, and/or breast tumors. (Refer to HTR2A gene.) With Htr2a knockout mutants in animal studies, chronic administration of antidepressant failed to produce antidepressant-like activity  large cohort with treatment outcomes and 2) large cohort with medication and genotyping. However, this study has some limitations that should be addressed. First, case controls were not matched. In our study, we corrected relatedness and population structure and adjusted for sex and age in the analyses to balance out possible confounders. Second, the two patient cohorts were significantly different in demographics and treatment history. Third, since the data were analyzed with various SSRIs, they did not allow analyses to focus on specific drugs. Fourth, this study is only focused on the HTR2A gene. Further studies of other of serotonin-related genes are required.This study applied gene-set association through analyses of pharmacogenetics association  and disease association  to identify if HTR2A SNPs that are associated with SSRI therapeutic phenotypes in both meta-analysis and mega-analysis from two MDD groups, but no association with MDD susceptibility was found. As expected for a single gene, the clinical impact of HTR2A on treatment outcome is modest. The power to detect loci with the observed effect sizes in our mega-analysis and meta-analysis is modest, ranging from 0.30–0.53 and 0.39–0.42, respectively, given a disease prevalence of 1–5%. Further replication studies in a larger sample are needed to confirm our findings and find other antidepressant therapeutic related genetic variants.In summary, our gene-based association analysis found 14 (out of 288) https://www.ebi.ac.uk/ena/data/view/PRJEB40684The datasets generated for this study can be found in EVA, C-FK designed analytical frameworks of data and performed data management and quality control and statistical analyses, wrote most parts of the paper, and revised the manuscript. Study conception and design: EL, AY, YY, and S-JT; acquisition of data: P-HK, Y-LL, and C-FK; analysis and interpretation of data: C-FK, P-HK, and S-JT; draft and revise manuscript: C-FK, P-HK, and S-JT. All authors read and approved the final manuscript.Genotyping of MDD subjects was funded by the RIKEN Center for Integrative Medical Science, Yokohama, Japan. This work was supported by grant MOST 109-2634-F-075-001 from Taiwan Ministry of Science and Technology, and grant V108D44-001-MY3-1 from the Taipei Veterans General Hospital. This work was financially supported (in part) by the Advanced Plant Biotechnology Center from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A group of pneumonia patients in Wuhan created an alarm worldwide by the end of December 2019. This emerging pneumonia soon became as a novel coronavirus (2019-nCoV or COVID-19) . It is tWe aimed to compute the relationship between the ABO blood type, age, sex, and ACE2 gene polymorphism with the susceptibility to COVID-19 in patients from Iran to test if the former can act as a biomarker for the latter.https://en.wikipedia.org/wiki/Blood_type_distribution_by_country). Then, statistical analysis was performed using SPSS software version 15 .Our study was performed on 94 random samples of dead cases infected with SARS-CoV-2. Of these, only 76 were identified by blood type. A recent survey of ABO blood group distribution of 80,982,137 normal people from Iran was used as comparison controls for patients with COVID-19 .Moreover, we analyzed the people’s age and sex as two other risk factors among patients with COVID-19 where the COVID-19 mortality rate for men was four times more than that of women. The genus of dead patients was 73 men and 21 women. These results corresponded to a significantly increased risk of men in comparison with women for COVID-19  (P< 0.00P= 0.004). A dead case with the “age of 94 is also shown in Also, current survey suggested that patients with ages of 50 < years are at greater risk relative to children who might be less likely to become infected or may show milder signs or even asymptomatic infection (https://en.wikipedia.org/wiki/Angiotensin-converting_enzyme_2). ACE2 is known as SARS-CoV-2 receptor and balance of ACE-ACE2 axis and blood pressure are important in patients with COVID-19 and susceptibility to the disease. Association between genotype of ACE2-ACE axis, blood group A, and blood pressure has been previously reported (“Angiotensin-converting enzyme 2 (ACE2) is an enzyme attached to the outer surface (cell membranes) of cells in the lungs, arteries, heart, kidney, and intestines “(reported , 6. Despreported , people


Several studies highlighted that obesity and diabetes reduce immune function. However, changes in the distribution of immunoglobins (Igs), including immunoglobulin-A (IgA), that have an important function in mucosal immunity in the intestinal tract, are unclear. This study aimed to investigate the impaired immune functions in the context of a diet-induced obese murine model via the assessment of the Igs in the intestinal villi. We used mice fed a high-fat diet (HFD) from four to 12 or 20 weeks of age. The distributions of IgA, IgM, and IgG1 were observed by immunohistochemistry. Interestingly, we observed that IgA was immunolocalized in many cells of the lamina propria and that immunopositive cells increased in mice aged 12 to 20 weeks. Notably, mice fed HFD showed a reduced number of IgA-immunopositive cells in the intestinal villi compared to those fed standard chow. Of note, the levels of IgM and IgG1 were also reduced in HFD fed mice. These results provide insights into the impaired mucosal immune function arising from diet-induced obesity and type 2 diabetes. Obesity and Type 2 diabetes mellitus (T2DM) have become quite prevalent worldwide; according to the World Health Organization (WHO), the global prevalence of obesity has nearly tripled since 1975 and is a major risk factor for diabetes and other non-communicable diseases ,4, chronThe intestinal tract has a simple columnar epithelium essential for nutrient uptake and is constantly exposed to commensals and exogenous bacteria, thus serving as an organ of the immune system with a well-developed immune function. Of note, the gut microbiome present in the gastrointestinal tract maintains a symbiotic relationship with the host, supporting metabolism and promoting the maturation of gut immunity . In addiThe gut microbiota is altered by the lifestyle and dietary choices of the host and is associated with obesity and T2DM, in addition to impaired immune function ,15,16,17It has been reported that promoting factors of IgA secretion are decreased in high-fat-diet (HFD)-fed mice . AnotherTo assess the appropriateness of our T2DM model, the body weight and casual blood glucose levels of mice were measured immediately before the experiments; the results are shown in p < 0.001), age (p < 0.001), and interaction (p = 0.003). Furthermore, the post hoc analysis indicated significant differences in the context of the diet in both 12-week-old mice  and 20-week-old mice , and in the context of age between 12- and 20-week-old mice fed with HFD ; post hoc analysis revealed SCD (131.0 ± 5.5 mg/dL) vs. HFD (157.0 ± 3.2 mg/dL) at 12 weeks (p = 0.008) and SCD (127.0 ± 7.9 mg/dL) vs. HFD (164.0 ± 2.5 mg/dL) at 20 weeks (p < 0.001) as significantly different groups  were compared among 12-week-old mice fed different diets a–e. Of nAdditionally, histological findings were compared among 20-week-old mice fed different diets as above . HE of 2p = 0.32) or diet models (p = 0.59) with respect to the areas of the LP considered (2) are shown in To disclose the differences in the distribution of Ig-immunopositive cells in the intestinal villi, we counted Ig-immunopositive cells in the entire or in specific areas of the LP. Overall, no significant differences were found between the different age groups (nsidered . All of p < 0.001) and diet models (p = 0.025). Furthermore, the post hoc analysis revealed significant differences between 12- and 20-week-old SCD-fed mice (p = 0.013) and between 12- and 20-week-old HFD-fed mice (p = 0.04), as shown in p = 0.017, p = 0.021, 12-week vs. 20-week in HFD; p = 0.030, HE-staining was used to assess all cells within the LP. The number of cells in the whole LP area was significantly different between the different age groups (p < 0.001) and diet models (p < 0.001); post hoc analysis demonstrated significant differences in the number of IgA-immunopositive cells between 12- and 20-week-old SCD-fed mice (p < 0.001) and between SCD-fed and HFD-fed 20-week-old mice (p < 0.001), as shown in p = 0.023; HFD vs. SCD in 20-week; p = 0.030, p = 0.031, HFD vs. SCD in 20-week; p = 0.003, p = 0.007, HFD vs. SCD in 20-week; p = 0.003, Additionally, at all ages and in the different diet models, the total numbers of Ig-immunopositive cells were higher with respect to IgA, followed by IgG1, and IgM. Of note, the number of Ig-immunopositive cells tended to be higher in SCD-fed mice than in HFD-fed mice. Importantly, the number of IgA-immunopositive cells in the whole LP area was significantly different between the age groups (p < 0.001). Furthermore, the post hoc analysis demonstrated significant differences in the number of IgM-immunopositive cells between 12-week-old HFD and SCD fed mice (p = 0.033) and between 20-week-old HFD and SCD-fed mice (p = 0.005), as shown in p = 0.003, p = 0.003, The number of IgM-immunopositive cells in the whole LP area was also significantly different between the diet models (p = 0.040) and diet models (p < 0.001); the post-hoc analysis demonstrated significant differences between 12- and 20-week-old SCD-fed mice (p = 0.017) and between 20-week-old SCD-fed and HFD-fed mice (p = 0.001), as indicated in p = 0.024, HFD vs. SCD in 20-week; p < 0.001, p = 0.016, HFD vs. SCD in 20-week; p = 0. 011, p = 0.041, Last but not least, the number of IgG1-immunopositive cells in the whole LP area was significantly different between age groups . Second, the method used was limited to paraffin-embedded tissue sections, which does not fully explain the changes in the number of Ig-positive cells in all of the villi spaces. In addition, we used a single mouse model, the obesity-induced T2DM model; therefore, the effects of non-caloric factors cannot be ruled out. Further studies are needed to provide detailed insight into the mechanism behind the decline of immune function in the context of obesity and diabetes. Moreover, we were not able to disclose whether the observed changes in Ig-positive cells were due to the high-fat diet originated from hyperglycemia and insulin excess (T2DM) or from excessive lipid intake (obesity). Last but not least, we were also not able to verify whether the underlying gut microbiome was synchronized with the changes in Ig-positive cells. Further studies are needed to clarify the above questions in the future. However, since the distribution of Ig-positive cells within the intestinal villi can be effectively reproduced by IVCT, the results of this study may represent a distinctive finding with biological relevance.Animal experiments were approved by the Health Science University Animal Care and Use Committee. C57Bl/6J mice  were housed within single cages in a ventilated incubator at the Health Science University under 12-h light/dark cycles (light on from 07:00 a.m. to 07:00 p.m.), and a temperature of approximately 23 ± 1 °C. Food and water were provided ad libitum. Of note, four-week-old male mice were assigned to one of the following four groups (n = 5 per group): group 1—mice fed SCD  from four to 12 weeks of age (12-week-old SCD); group 2—mice fed SCD from four to 20 weeks of age (20-week-old SCD); group 3—mice fed HFD  from four to 12 weeks of age (12-week-old HFD); and group 4—mice fed HFD from four to 20 weeks of age (20-week-old HFD). Immediately prior to the experiments, the body weight and casual blood glucose levels of the mice were measured to confirm obesity and diabetes. The casual blood glucose levels were measured with blood from the tail vein using a blood glucose meter .Mice were anesthetized via injection of a mixture of medetomidine hydrochloride , midazolam , and butorphanol tartrate  at a dosage of 0.1 mL/10 g body weight. Of note, fasting was not performed to effect on immunoglobulin localization in the intestinal tract. Small intestine tissues, 2–3 cm from the end of the stomach, were exposed outside the body under normal blood circulation and carefully put on aluminum sheets in their abdominal cavities. Since artifacts usually appear farther away from the contact point of the cryogen in IVCT, the luminal side of the jejunum was exposed by an incision along the long axis of the intestine. IVCT was immediately performed via direct pouring of isopentane–propane cryogen (−193 °C), precooled in liquid nitrogen, over the small intestines from the luminal side. Liquid nitrogen was additionally poured over the frozen small intestines, which were then carefully taken out. Of note, IVCT in the context of the intestine was carried out as previously reported .The freeze-substitution solution consisted of absolute acetone containing 2% paraformaldehyde. The frozen small intestine tissues were freeze-substituted at approximately −80 °C in dry ice-acetone for 48 h and then put into a freezer at −30 °C for 2 h and at −10 °C for 2 h. Tissues were then refrigerated at 4 °C for 2 h and finally kept at room temperature for 1 h. Finally, tissues were washed with pure acetone, immersed in xylene, and routinely embedded in common paraffin wax.All paraffin-embedded tissues were cut into 5-μm thick serial paraffin sections and mounted on Matsunami Adhesive Slide coated glass slides . The thin sections were de-paraffinized with xylene and rehydrated in a graded series of ethanol and distilled water. Some sections were routinely stained with HE to observe the tissue morphology in the context of frozen conditions using a light microscope. After that, the sections were processed for immunohistochemistry analyses and incubated with 0.3% hydrogen peroxide in phosphate-buffered saline (PBS) and 2% gelatin in PBS for 1 h each. The sections were then immunostained via incubation overnight with different primary antibodies in 2% gelatin  at 4 °C. The primary antibodies used were goat or rabbit polyclonal antibodies against murine IgA , IgM , and IgG1 . The immunocontrols were thin sections incubated in 2% gelatin without primary antibodies. Then, the immunostained sections were incubated with horseradish peroxidase-conjugated donkey anti-goat  for IgA and -IgG1 or goat anti-rabbit IgG  for IgM, at room temperature for 1 h and finally visualized with diaminobenzidine (DAB) in a buffer solution containing hydrogen peroxide for 3 or 5 min. The sections were observed as optical microscopic images under an all-in-one microscope . Of note, the development of brownish cellular colors after reaction with DAB was defined as immunopositivity.https://fiji.sc/wiki/index.php/Fiji), and the LP were manually traced. In addition, to verify the differences in the distribution of cells within the villi, they were divided into three sections using the following procedure. First, two points where the simple column epithelium on both sides of the villi failed  were connected to obtain a baseline. Second, the point of intersection of the villi with the parallel line to the baseline, furthest from the baseline, was set as the tip. Third, the line connecting the tip and the midpoint of the baseline was set as the axis. Finally, the axis was divided into three parts, top, middle, and bottom from the tip. This method is based on that used in a previously published study in which the chorionic villi were divided into three sections [2, either considering the whole LP, or divided sections.The distribution of Igs in the villi of the jejunum was assessed based on the quantification of cells in the optical microscopy images. The jejunal villi were determined to be centrally located when a single layer of columnar epithelium and central lacteal vessels were observed, and cells in the LP were counted. The images obtained were processed using ImageJ and Fiji plugins .Statistical analysis was performed using the two-way ANOVA followed by Holm-Šídák’s post hoc analysis, because there were four groups with different ages (12 and 20 weeks) and diet (SCD and HFD). The GraphPad Prism  was used to conduct statistical analyses. A In the present study, tissue fixation with IVCT confirmed the distribution and structure of Ig-positive cells in the context of the “living animal morphology of small intestinal villi” in SCD- and HFD-fed mice. The results suggest that IgA normally increases in the LP  of SCD-fed mice between the ages of 12 and 20 weeks. However, HFD feeding suppresses the increase in IgA-immunopositive cells in the intestinal villi. Altogether, our results provide insights into the impaired mucosal immune function in diet-induced obesity and T2DM from the biological perspective.


RNA interference represents one of the most appealing therapeutic modalities for cancer because of its potency, versatility, and modularity. Because the mechanism is catalytic and affects the expression of disease-causing antigens at the post-transcriptional level, only small amounts of therapeutic need to be delivered to the target in order to exert a robust therapeutic effect. RNA interference is also advantageous over other treatment modalities, such as monoclonal antibodies or small molecules, because it has a much broader array of druggable targets. Finally, the complementarity of the genetic code gives us the opportunity to design RNAi therapeutics using computational, rational approaches. Previously, we developed and tested an RNAi-targeted therapeutic, termed MN-anti-miR10b, which was designed to inhibit the critical driver of metastasis and metastatic colonization, miRNA-10b. We showed in animal models of metastatic breast cancer that MN-anti-miR10b accumulated into tumors and metastases in the lymph nodes, lungs, and bone, following simple intravenous injection. We also found that treatment incorporating MN-anti-miR10b was effective at inhibiting the emergence of metastases and could regress already established metastases in the lymph nodes, lungs, and bone. In the present study, we extend the application of MN-anti-miR10b to a model of breast cancer metastatic to the brain. We demonstrate delivery to the metastatic lesions and obtain evidence of a therapeutic effect manifested as inhibition of metastatic progression. This investigation represents an additional step towards translating similar RNAi-targeted therapeutics for the systemic treatment of metastatic disease. Previously, we developed a therapeutic that could broadly target elements of the cellular RNAi apparatus in tumors and metastases, following simple intravenous injection. These included small interfering RNAs, antagomirs for the suppression of endogenous microRNAs, and microRNA mimicsTo shuttle therapeutic oligonucleotides to tumors and metastases, we employed dextran-coated iron oxide nanoparticles optimized in terms of size, charge, and surface coating in order to ensure stability in the circulation, a long circulation half-life, effective accumulation into the interstitium of primary and metastatic tumor lesions through hemodynamic mechanisms, and uptake of therapeutically significant amounts by tumor cells, through endocytosis. While unproven, mechanisms related to “metabolic” targeting of the dextran-coated nanoparticles through the Warburg effect were also hypothesized. The nanoparticles were linked to oligonucleotides by finetuning their surface coating in order to be able to chemically conjugate large amounts of oligo that, while retained onto the nanoparticle surface in the circulation and interstitium, is separated from the nanoparticles in the reducing intracellular environment and is free to enter the RNA induced silencing complex (RISC).8. MicroRNA-10b is one of the best validated targets in metastasis that has been shown to drive metastatic dissemination10 and metastatic cell viability5. Multiple clinical studies have proven the value of miR-10b as a biomarker of metastasis in patients with a variety of malignancies, including breast, hepatocellular, lung, pancreatic, colorectal, gastric, ovarian adenocarcinoma, and melanoma, to name a few. MircoRNA-10b has been validated as a biomarker of clinical parameters, such as stage, presence of metastases, relapse-free survival, overall survival, degree of invasion, and time to recurrence (reviewed in11).The majority of our work employed an antagomir-based strategy that could effectively inhibit microRNA-10b in primary tumors and metastases from breast cancer8. These studies came on the heels of earlier seminal work that established miR-10b as a critical effector of tumor cell migration and invasion and metastatic initiation12. Combined, the roles of microRNA-10b in metastatic initiation and progression established its importance as a therapeutic target in cancer. For that reason, a major goal of our work has been to develop and test therapeutics based on miR-10b inhibition.Our focus on microRNA-10b was motivated not only by its proven clinical relevance as a biomarker of metastatic progression but also by our own discovery that miR-10b is a major driver of metastatic cell viability13. We performed thorough mechanistic studies elucidating the biological effects of MN-anti-miR10b6 and showed that MN-anti-miR10b affects molecular processes downstream of E-cadherin and, through its effect on HOXD10 and c-JUN profoundly inhibits the capacity of tumor cells to migrate and invade surrounding tissue and induces apoptosis in metastatic tumor cells. In this earlier work, we tested the effectiveness of the therapeutic in murine models of triple negative breast cancer. We confirmed uptake by primary and metastatic tumor cells, found in the lymph nodes, lungs, and bone, following intravenous injection8. When treatment with MN-anti-miR10b was initiated prior to the formation of overt metastases, metastasis was prevented3. When treatment was initiated after the formation of detectable metastases, therapy involving MN-anti-miR10b elicited durable regressions with no evidence of systemic toxicity8. Importantly, after four to six weekly treatments with MN-anti-miR10b in combination with low dose chemotherapy, we observed complete regression of detectable metastatic lesions. At that point, even though therapy was discontinued, metastases did not recur8.To inhibit miR-10b, we used a therapeutic (termed MN-anti-miR10b), which consisted of LNA-incorporating antagomirs conjugated to dextran-coated iron nanoparticles8. In the present study, we focused more distinctly on the capacity of MN-anti-miR10b to target brain metastatic lesions from breast cancer and to elicit a therapeutic response. We used the MDA-MB-231-Br “brain homing” subclone of the human MDA-MB-231 cell line, which represents a pure clonal population of brain-tropic metastatic cells and therefore permits directed study of therapeutic response in the brain lesions.In our earlier work, we obtained evidence of MN-anti-miR10b uptake by micrometastatic lesions in the brains of mice implanted orthotopically with the highly aggressive 4T1 murine breast cancer cell line4. The nanoparticles were labeled with near infra-red dye, Cy5.5, and conjugated to the heterobifunctional linker N-succinimidyl 3-[2-pyridyldithio]-propionate  and activated oligos sequentially. Briefly, Cy5.5-NHS was conjugated to NP to produce Cy5.5-labeled magnetic nanoparticles (MN) overnight. SPDP in anhydrous DMSO was incubated with MN. The anti-miRNA-10b LNA antagomir was modified with the 5′-Thiol-Modifier C6 disulfide (5′-ThioMC6), which was utilized for conjugation to SPDP-modified MN. The disulfide on the oligonucleotide was activated by 3% Tris (2-carboxyethyl) phosphine hydrochloride , followed by purification with ammonium acetate/ethanol precipitation treatment prior to conjugation to SPDP-modified MN. After TCEP activation and purification, the oligo was dissolved in water and incubated with the SPDP-modified MN overnight. MN-anti–miR-10b was freshly prepared each week.Aminated dextran coated iron oxide nanoparticles (NP) were synthesized using a protocol similar to ones published previously−1 cm−1). The concentration of MN was obtained by iron assay . The size of MN was measured using transmission electron microscopy  in order to determine the diameter of the iron-oxide core and dynamic light scattering  in order to determine the hydrodynamic (Z-average) diameter, as described previously4.The number of oligos per MN was quantified by gel electrophoresis after the treatment with TCEP. The number of Cy5.5 was determined by UV/VIS spectrophotometry  as recommended by the provider14.Human brain-tropic MDA-MB-231-BrM2-831 cells were kindly provided by Dr. Joan Massagué . The cells are transduced with a lentivirus expressing a triple-fusion reporter encoding herpes simplex virus thymidine kinase 1, green fluorescent protein (GFP) and firefly luciferase (Luc). The cells were cultured in DMEM supplemented with 6 cells in 100 mL HBSS)15. Briefly, after cleaning the skin over the chest, a needle was inserted 2 mm to the left of the sternum and 12 mm above the tip of the xiphoid process. The tip of the needle was slightly in the caudal direction and was angled 25° relative to the sagittal plane16. Since the cells express green fluorescent protein (GFP) and luciferase (Luc), they can be detected by noninvasive optical imaging for the quantification of metastatic tumor burden. All animals were scanned weekly in order to monitor tumor growth and metastasis in the brain. Finally, MN-anti-miR-10b was administered after the confirmation of breast cancer metastases in the brain and continued every week following the pre-fixed schedule8. Animals were sedated under Isoflurane anesthesia 10 min before cardiac cell injection, peritoneal injection or imaging. For post-procedural pain relief, animals were administered buprenorphine twice daily for the duration of the experiment . All procedures were carried out in aseptic fashion in clean facilities. Animals were observed daily for their level of activity and normal eating, drinking and grooming behavior. According to AVMA guidelines, a high dose of sodium pentobarbital was injected intraperitoneally (200 mg/kg IP) to sacrifice animals at the end of study. Animals were housed in the MGH Animal Facility, which is under the supervision of the MGH Office of Laboratory Animal Research. All animal experiments were performed in compliance with institutional guidelines and approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital .Ten female Balb/c nude mice  were directly injected into the heart with MDA-MB-231-BrM2-831 cells . Anesthetized mice were injected i.v. with Cy5.5 labeled MN-anti-miR10b  and scanned by fluorescence reflectance imaging 24 h later. The acquisition conditions are summarized as follows: Exposure time, 0.5 s; Binning factor, 8; Excitation filter range, 675 nm; Emission filter range, 720 nm; f number, 2. Tumor location and growth were monitored by fluorescence reflectance imaging using filters for green fluorescence protein (GFP). The acquisition conditions were: Exposure time 4 s; Binning factor, 8; Excitation filter range, 465 nm; Emission filter range, 540 nm; f number, 2. All images were analyzed using the Living Image Software . Radiant efficiency of the excised tissues was used for signal quantificationd-luciferin potassium salt in DPBS  12 min before image acquisition. Identical image acquisition settings  and the same ROI were used to obtain total radiance (photons/s) over the ROI. BLI was performed for about 6–15 min to obtain the maximum radiance. All images were processed using the Living Image Software . Total flux from the bioluminescence readings was used for signal quantification5.BLI was used to evaluate tumor burden. Imaging was performed using the IVIS Spectrum imaging system. Anesthetized mice were injected intraperitoneally with Treatment was delivered to animals immediately after the confirmation of breast cancer metastases in the brain by BLI. The treatment protocol consisted of injection of MN-anti-miR-10b or MN nanoparticles without antagomir intravenously  every week. Treatment was administered weekly for 3 weeks and was stopped to analyze the distribution of breast cancer metastases and the uptake of MN-anti–miR-10b in breast cancer metastases in the brain.Excised tissues were embedded in Tissue-Tek OCT compound  and snap frozen in liquid nitrogen. The tissues were cut into 7 µm sections and fixed in 4% formaldehyde for 10 min. The slides were analyzed using a Nikon E400 fluorescence microscope , equipped with the necessary filter sets . Images were acquired using a charge coupled device camera with near-IR sensitivity. The images were analyzed using ImageJ .For the HOXD10 staining, tissues were incubated with recombinant anti-HOXD10 antibody [EPR9374] (ab138508), followed by fluorescently-labeled anti-rabbit secondary polyclonal antiserum . The slides were analyzed by fluorescence microscopy as described above.p < 0.05 was considered statistically significant.Data were expressed as mean ± SD. Statistical comparisons were made using a two-tailed t-test . A value of 13. The number of amino groups per NP was determined as 74. A near infra-red fluorescent dye, Cy5.5 was loaded onto NP, to generate MN. The ratio of Cy5.5 per MN was found to be 2.9 ± 0.4. Following conjugation to the anti-miR10b antagomir to generate MN-anti-miR10b, the number of antagomirs per MN was determined as 7.5 ± 0.5  to introduce amino groups onto the surface of the nanoparticles and functionalize them with a near infrared fluorescent dye (Cy5.5) to generate the magnetic nanoparticle template, MN. Anti-miR10b antagomirs were conjugated to MN to produce MN-anti-miR10b, using a protocol similar to ones previously described15. To generate metastases to the brain, MDA-MB-231-BrM2-831 cells were administered by direct cardiac injection. One week after cell injection, all mice were monitored in order to confirm the formation of metastases. Brain metastases were clearly visible by BLI 1 week after cell injection. At that point, MN-anti-miR10b was injected intravenously. Twenty four hours later, the animals were imaged by noninvasive bioluminescence and fluorescence optical imaging with filters appropriate for Cy5.5. As seen in Fig. The MDA-MB-231-BrM2-831 breast cancer cell line is transduced with a lentivirus expressing a triple-fusion reporter. The reporter encodes herpes simplex virus thymidine kinase 1 suitable for detection by nuclear imaging, green fluorescent protein (GFP) optimal for detection by microscopy, and firefly luciferase (Luc) optimal for detection by bioluminescence optical imaging (BLI)Accumulation of MN-anti-miR10b in the brain metastatic lesions was confirmed by fluorescence microscopy. We observed perivascular micrometastatic lesions from the GFP-expressing cell line, which also accumulated MN-anti-miR10b Fig. c.12 and is commonly used to demonstrate target engagement, following treatment with miR-10b inhibitors. Consistent with the known mechanism of action of miR-10b, inhibition of the target by MN-anti-miR10b upregulated HOXD10 in brain metastases  by immunofluorescence. HOXD10 is a known miR-10b target, which demonstrates an inverse correlation with miR-10b expression5, the animals with BLI-detectable brain metastases were injected weekly with MN-anti-miR10b, applied as monotherapy. Treatment was continued for an average of 3 weeks, at which point outgrowth of the metastases in the control animals necessitated termination of experiments. Metastatic burden was measured by bioluminescence imaging. Reduction in metastatic burden in the brain was detectable even after the first treatment  is likely proportional to the rate of cell division in the metastatic lesions. The cell line used in the present study and the cell line used in our prior studies on lymph-node metastatic breast cancer are both MDA-MB-231 sublines with comparable growth kinetics. However, the dose of the therapeutic used in the current study as monotherapy was double the dose used in the prior investigation, in which combination with a cytostatic was necessary to induce regression5. While this is not a controlled side-by-side comparison, it implies that MN-anti-miR10b could be effective as monotherapy at higher doses, optimized for the growth kinetics of the metastatic lesions.A key point that needs to be addressed has to do with the feasibility of using MN-anti-miR10b as monotherapy against breast cancer metastatic to the brain. In our prior studies, at lower doses, MN-anti-miR10b was very effective at preventing the emergence of detectable macrometastatic disease in a model of lymph-node metastatic breast cancer and could induce arrest of metastatic progressionIn summary, we are presenting new evidence of the utility of our miR-10b targeted therapeutic, MN-anti-miR10b, for the treatment of metastatic breast cancer. We further validate the fundamental and unique value of miRNA-10b as a therapeutic target for the tangible regression of established metastases. This, in addition to similar effective and clinically-relevant vehicles, represents a modality for cancer treatment that is radically different from other modern therapies, such as monoclonal antibodies and small molecules, both in terms of the underlying molecular biology and the potential for rational delivery to target organs.